JPH02242678A - Novel beta-mannosidase and production thereof - Google Patents

Abstract

PURPOSE:To hydrolyze beta-D-mannan and enable efficient recovery and utilization of a hydrolyzate by culturing an AS-440 strain belonging to the genus Bacillus and using the resultant enzyme having specific physico-chemical characteristics. CONSTITUTION:A microorganism AS-440 (FERM P-8860) capable of producing intracellular beta-mannosidase is inoculated into a culture medium, containing a carbon source, such as 'KONJAK' (devil's-tongue) powder, a nitrogen source, such as peptone, inorganic salts and minor nutriments, regulated to pH 7.5-11.5 with 0.5% sodium hydrogencarbonate and aerobically cultured at 30-40 deg.C. Microbial cells in the culture solution are then collected by a well-known means, such as centrifugation or filtration. The resultant microbial cells can be directly used. Alternatively, the microbial cells can be crushed, extracted and then purified by a normal method for purifying the enzyme. Thereby, beta-D-mannan can be hydrolyzed in a simple and economical step with a high efficiency and the unnecessary mannan can be rapidly removed for the objective hydrolyzate can be mass-produced.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a novel β-mannosidase and a method for producing the same. More specifically, the intracellular β-mannosidase, which has an optimum pH for enzymatic reaction in the vicinity of neutrality, obtained by culturing a new alkaliphilic microorganism belonging to the genus Bacillus and has an optimum pH for growth on the alkaline side; It concerns the manufacturing method.

Conventional technology β-mannosidase is a low-molecular β-D-mannan (mannan, glucomannan, galactomannan, galactoglucomannan) that has a β-mannosidic bond in the molecule.
It is an enzyme that hydrolyzes mannoside bonds sequentially from the non-reducing end site to produce mannose.

Aipori nuts (scientific name: Phytelephas macrocarba) and corozo are well known as those containing β-D-mannan. Plants containing β-1°4-mannan in this pond include Phoenicus canariensis and Orchis maculata, which belong to the palm family.

Typical examples of galactomannan are the mucilages contained in carob and guar seeds, locust bean gum, and guar gum. It is used.

Galactomannan is also found in large amounts in leguminous plants such as soybeans, coffee beans, alfalfa, red clover, and fenugreek. Other galactomannan-containing plants include Genista scoparia, Bredeirasha
Known examples include Fuerox and Leucaena glauca.

Konjac (scientific name: Nearmorphopharus konjac) is the most famous glucomannan-containing substance.
Known examples include arum roots of the Cytomaceae family, jackpines of the Pinus genus, bulbs of the Orchidaceae family, and plants of the spruce genus such as rainbow pine and harem. Other glucomannan-containing plants include Asparagus officinalis, Eremurus
Ruscus, Eremurus legeri, Eremurus spectabilis, Phaseolus aureus, etc. are known. Glucomannan is obtained from these plants by an alkali extraction method or the like. Further, these β-D-mannans are consumed in large quantities industrially in the food industry and the textile industry as thickeners or thickeners.

β-mannosidase, which has been known as an enzyme that hydrolyzes β-D-mannan into mannose units from the non-reducing end, has been developed in animals [biochemistry (f3io
Chemistry), 1972. 11. 14
93-1501: Biosimica e Biophysica
Acta (Biochim, Biophys, Acta)
, 1973.268, 488-496: Biochim,
Biophys, Acta), 1973, 315.1
23-127), plants [Journal of Biological Chemistry - (J, Biol, Che
m, ), 1964. 239. 990-992)
, microorganisms [biostain force
(Biochim, Biophys, Acta),
1978, 522.521-530: Japanese Patent Publication No. 51-38
No. 486)] and other enzymes have been well studied.

However, all of these enzymes have low productivity 2 and many require complicated culture and purification methods, leaving difficulties in using the enzymes industrially at low cost.

Problems to be solved by the invention β-D- exists in large quantities as a renewable resource in the natural world.
In order to effectively utilize mannan, especially to efficiently recover and utilize mannooligosaccharides and sugars such as mannose, glucose, and galactose through enzymatic hydrolysis of mannan, it is important that the enzyme has excellent stability and is easy to purify. preferable.

However, as mentioned above, the previously proposed β-mannosidases, which have various origins such as animals, plants, and microorganisms, are insufficient in terms of productivity, and their production and purification methods are also complicated. It was still unsatisfactory for practical use.

Therefore, it is important to newly develop this type of enzyme that is easy to produce and purify and has high stability as described above.
-It is of great significance in decomposing mannan or recovering and utilizing decomposition products (mannose, etc.).

Therefore, the first object of the present invention is to provide a novel enzyme, β-mannosidase, that satisfies the above various requirements.

A second object of the present invention is to provide a method for producing the novel β-mannosidase described above using a novel microorganism that can produce the enzyme easily and in high yield.

Means for Solving the Problems The present inventors extensively searched the natural world to obtain microorganisms capable of producing enzymes having the above-mentioned properties that β-mannosidase for industrial use should have. result,
The present invention was completed by discovering that bacteria belonging to the genus Bacillus, which has an alkaline optimum pH for growth, can produce β-mannosidase that meets the above requirements and can produce it with good productivity.

The novel β-mannosidase provided by the first aspect of the present invention has the following physical and chemical properties: (a) Action: Hydrolyzes β-mannosidic bonds sequentially from the non-reducing end,
Produces mannose.

(b) Substrate specificity: Completely decomposes β-methyl(ethyl)-D-mannoside, and also acts on β-linked mannose-containing oligosaccharides to liberate mannose.

β-D mannoside of p-nitro-phenyl-glycoside can be used as a substrate, but α-D mannoside, α-D-curcoside, β-D-glucoside, α-D-galactoside,
β-D galactoside, β-D-xyloside, αL-fucoside, and β-D-glucuronide cannot be used as substrates.

(c) Optimal pH and stable pH range: The optimal pH is 6 to 7, and is stable within the pH range of 6 to 9 under heating conditions at 40° C. for 30 minutes.

(d) Stability against temperature: Stable up to 45°C under conditions of pH 6, 5, and heating for 30 minutes.

(e) Range of suitable temperature for action: It has an optimum operating temperature around 50°C.

(f) Inactivation conditions: pH 5.0 and I under treatment conditions of 40°C and 30 minutes.
It is completely inactivated by O. Further, in the treatment of ρ■6.5.30 minutes, the activity is completely inactivated at 55°C.

(g) Molecular weight by gel filtration method: 63,000±3.000 The above novel β-mannosidase can be produced by the production method provided by the second aspect of the present invention. The method of the present invention provides the above-mentioned β belonging to the genus Bacillus.
- It can be obtained by a method characterized by culturing a microorganism that intracellularly produces mannosidase, collecting the microorganisms, and then isolating and purifying the microorganisms.

The novel intracellular β-mannosidase-producing bacterial strain used in the method of the present invention was newly searched for and isolated from nature by the present inventors. These strains were listed in Bergey's Manual of Determinative Bacteriology.
terminative Bacteriology)
, 8th edition and The Genus Bacillus (The Ge
nusBacillus, Department of Agriculture, USA
According to the Bacillus genus, it is an aerobic sporobacillus, is motile, has periflagella, and has a positive Durham stain or a positive variable bnopecatalase test. It was clear that the strain belonged to the genus Bacillus, but since it grows well in an alkaline pH range of 7.5 to 11.5, it was considered to be a new strain that is taxonomically superior to the known Bacillus genus bacteria.

Table 1 below shows various mycological properties of isolated intracellular β-mannosidase producing cells.

Table 1 (1) Table 1 (2) Table 1 (3): No growth or negative In addition, the above bacteria should be submitted to the Institute of Microbiology, Agency of Industrial Science and Technology by FER.
It has been deposited as MP-8860 (AS-440).

Next, the novel method for producing intracellular β-mannosidase of the present invention will be explained in more detail.

The above-mentioned intracellular β-mannosidase producing microorganisms are inoculated into a suitable medium and cultured aerobically for 48 to 72 hours at 30 to 40° C. from the viewpoint of the growth temperature of the microbial cells. Here, the medium contains inorganic salts and micronutrients as necessary in addition to a carbon source and a nitrogen source.

First, various conventionally known materials can be used as the carbon source, and typical examples include konjac flour, locust bean gum, carob gum, guar gum, and plants containing these.

There are also no particular restrictions on nitrogen sources; organic nitrogen such as yeast extract, peptone, meat extract, corn stay 7' IJ car, amino acid solution, soybean meal, or inorganic nitrogen such as ammonium sulfate, urea, ammonium nitrate, ammonium chloride, etc. For example, these methods are inexpensive and easy to handle.

It goes without saying that the organic nitrogen source serves as a carbon source. Furthermore, in addition to such carbon sources and nitrogen sources, it is also possible to add various commonly used salts, such as inorganic salts such as magnesium salts, potassium salts, phosphates, and iron salts, and vitamins. .

Suitable media for use in the method of the invention include, for example, 1% konjac flour, 2% polypeptone, 0.2%
yeast extract, 0.1% 2HPO.

and 0.2% Mg5O<-7H20.

Furthermore, since the growth pli of the microorganisms used in the method of the present invention is within the basic range, it is necessary to adjust the pH value of the medium using an appropriate alkali. 0.5 for that
% sodium bicarbonate can be cited as a typical example, but alkaline reagents such as sodium hydroxide, potassium hydroxide, sodium carbonate, sodium phosphate, calcium hydroxide can also be used, but are not limited thereto.

The bacteria used in the method of the present invention produce β-mannosidase within their cells and accumulate it there. Cultivation of these bacteria can be carried out either batchwise or continuously, and the produced enzymes can be separated and purified, for example, as follows.

That is, it is possible to first collect the bacterial cells in the culture solution by known means such as centrifugation or filtration, and then use the obtained bacterial cells as they are for the hydrolysis reaction of mannooligosaccharides, which is economical. It is advantageous.

Of course, it can also be used after further purification.

For this purpose, for example, after bacterial cell crushing and extraction, salting out with ammonium sulfate, solvent precipitation with ethanol noveacetone, isopropanol, etc., ultrafiltration, gel filtration, general enzyme purification using ion exchange resin, etc. Can be purified.

Below, one example of a preferred purification method for β-mannosidase of the present invention will be explained.

The above AS-440 strain belonging to the alkaliphilic Bacillus genus is inoculated into, for example, the above medium, and incubated at 37°C for 4 hours.
The culture solution obtained by culturing aerobically for 8 hours was heated to 12,00
Cells are collected by centrifugation at 0° C. for 30 minutes to obtain cells weighing 10 g wet. Next, the bacterial cells were cooled in ice water and added to 10 mM phosphate buffer (pH 7.0.
) and ultrasonic crushing is carried out several times for a total of about 3 minutes. Then, at 12.00 Or, p, m, 30 at 0°C.
Centrifuge for 1 minute to remove the residue and obtain 50 ml of supernatant.

Next, ammonium sulfate was added to this supernatant to give a concentration of 75%.
Bring to saturation and leave overnight at 4°C. Filter the resulting precipitate,
Dissolved in 10mM phosphate buffer (pH 7.0) - night 4
Dialyze against the same buffer at °C.

The resulting precipitate was removed by centrifugation, and the resulting supernatant was adsorbed onto DEAD-)Yopal 650M equilibrated with the above buffer, followed by a concentration gradient of the above buffer containing 0.1 to 0.5 M NaCl. The enzyme is eluted by a method.

The eluted active fractions were collected and incubated against the same buffer overnight for 4 hours.
After dialysis at °C, it is adsorbed onto hydroxyapatite equilibrated with the same buffer. Next, the enzyme is eluted with 0.4 M'J acid buffer (pH 8,0), and the active fractions are collected and concentrated using an ultrafiltration membrane with an average molecular weight cutoff of 10.000. The concentrated enzyme is a column for high-performance liquid chromatography for protein separation and purification.
HODBX protein)l! Is-2003
Filled with 10mM! Elute using J acid buffer (pH 7,0). After concentrating the active fraction thus obtained, it was subjected to chromatography again under the same conditions using the same blade ram, and the obtained active fraction was concentrated and subjected to polyacrylamide gel disk electrophoresis [Annals New York Academic Science (^ NN, N, Y,
Acad, Sci.), 121.404 (1964
)] to obtain 15 mg of a homogeneous enzyme preparation. The activity yield was 18%.

The method for measuring β-mannosidase activity and the method for displaying the activity are as follows.

i.e. 0.2M phosphate buffer <pl(7,0) 0.2
Mix 1 ml of enzyme solution Q with 2 ml of 3 mM p-nitrophenyl-β-D-mannopyranoside aqueous solution Q, react at 40°C for 10 minutes, and then add 1.0 ml of 0.5 M sodium carbonate aqueous solution. After inactivating the enzyme, add water to make 3 ml. The degree of coloring is measured using ultraviolet light (wavelength 42
011m) with 1 μmol/ml p-nitrophenol as a standard.

The unit of enzyme activity is 1 μmol per minute under the above conditions.
The amount of enzyme that liberates p-nitrophenol is expressed as one unit.

The molecular weight of β-mannosidase obtained by the method of the present invention is 63.000±3.000. Note that this molecular weight was determined by gel filtration method.

Table 2 shows a comparison of the physicochemical properties and enzymatic chemical properties of the β-mannosidase of the present invention and the conventionally known β-mannosidase derived from microorganisms.

β-D-mannan is contained in relatively large amounts in various plants, and is used industrially in various fields as a glue, thickener, and food material, either as it is or after chemical modification. It is used in many ways.

When this β-D-mannan is used, for example, as a thickening agent in the textile industry, it is removed after a certain processing process is completed, and in that case, its degrading enzyme, β-mannosidase, etc. are generally used. This type of enzyme is also used when β-D-mannan is hydrolyzed and the resulting decomposition product is used.

However, all of the conventionally known β-mannosidases have low productivity and many require complicated culture and purification methods. Therefore, it is expensive and difficult to use on a large scale industrially as described above.

Furthermore, the extraction process of β-D-mannan is generally carried out in an alkaline environment, and in such cases it is advantageous to use an enzyme that is more stable than known enzymes in alkaline conditions and has a higher optimum pH. be. In other words, with conventional enzymes that have an optimum pH on the acidic side, the extract is treated with a neutralizer before the decomposition reaction.
It is necessary to adjust the pH, which not only complicates the process but also increases cost.

Therefore, there is a need to develop highly productive methods, and there is a need to develop enzymes with a higher optimal p++ than known enzymes.

According to the specific microorganisms discovered by the present inventors, the above β-
It is possible to obtain a large amount of an enzyme that satisfies all the requirements related to the decomposition of D-mannan, and various conventional problems can be solved at once.

That is, by using the parent strain newly discovered according to the present invention, it can be obtained in large quantities and at low cost by a simple method, so that the problems of mass production and cost can be overcome. Therefore, large-scale industrial use becomes possible.

In addition, the optimal p in the mannan decomposition reaction of the obtained enzyme is
Since H is near neutrality, the next decomposition reaction can be started immediately after the mannan extraction process and slight pH adjustment. Therefore, the disassembly operation is simplified and economically advantageous.

Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example FERMP-886 to Institute of Microbial Technology, Agency of Industrial Science and Technology
Alkaliphilic bacterium Bacillus deposited as 0 ^S-0
44 strains were placed in a 500 m Erlenmeyer flask with 2% palm oil residue, 1% soybean residue, 30.2% KNO, and Na2HP.
O40.1%, MgSO,・7) 1200.02%
and 100ml of culture solution containing 0.5% soda carbonate (
Inoculated into pl (10, O) and incubated at 40℃ for 50 hours at 25Or
, 21 m, and cultured with shaking. Next, this cell suspension was heated to 12.00 Or, plm.

After centrifugation at 0°C for 30 minutes, the β-mannosidase activity of the resulting supernatant was measured and found to be 38 units/ml.

Effects of the Invention As described in detail above, the present invention provides one of the β-D-mannan hydrolyzing enzymes, namely β-mannosidase, which has an optimum pH for enzymatic reaction on the alkaline side. By using it, β-D-mannan can be decomposed with high efficiency in a simple and economical process, unnecessary mannan can be quickly removed, or the desired decomposition product can be produced. .

Moreover, according to the method for producing β-mannosidase of the present invention, the enzyme can be easily obtained with high productivity. Therefore, it becomes possible to utilize the enzyme at low cost on an industrial scale.

Claims (6)

[Claims] (1) A novel β-mannosidase having the following physical and chemical properties: (a) Action: Hydrolyzes β-mannosidic bonds sequentially from the non-reducing end to generate mannose. (b) Substrate specificity: Completely decomposes β-methyl(ethyl)-D-mannoside, and also acts on β-linked mannose-containing oligosaccharides to liberate mannose. β-D-mannoside of p-nitro-phenyl-glycoside can be used as a substrate, but α-D-mannoside, α-D-glucoside, β-D-glucoside, α-D
- Galactoside, β-D-galactoside, β-D-xyloside, α-L-fucoside, and β-D-glucuronide cannot be used as substrates. (c) Optimal pH and stable pH range: The optimal pH is 6 to 7, and is stable within the pH range of 6 to 9 under heating conditions at 40° C. for 30 minutes. (d) Stability against temperature: Stable up to 45°C under conditions of pH 6.5 and heating for 30 minutes. (e) Range of optimum temperature for action: The optimum temperature for action is around 50°C. (f) Inactivation conditions: pH 5.0 and 1 under treatment conditions of 40°C and 30 minutes.
At 0, it is completely inactivated. Furthermore, when treated at pH 6.5 for 30 minutes, the activity is completely inactivated at 55°C. (g) Molecular weight by gel filtration method: 63,000±3,000 (2) FERMP-
The β-mannosidase according to claim 1, which is produced by a bacterium deposited as 8860 (AS-440). (3) The following physical and chemical properties: (a) Action: Hydrolyzes β-mannoside bonds sequentially from the non-reducing end to produce mannose. (b) Substrate specificity: Completely decomposes β-methyl(ethyl)-D-mannoside, and also acts on β-linked mannose-containing oligosaccharides to liberate mannose. β-D-mannoside of p-nitro-phenyl-glycoside can be used as a substrate, but α-D-mannoside, α-D-
glucoside, β-D-glucoside, α-D-galactoside, β-D-galactoside, β-D-xyloside, α-
L-fucoside and β-D-glucuronide cannot be used as substrates. (c) Optimal pH and stable pH range: The optimal pH is 6 to 7, and is stable within the pH range of 6 to 9 under heating conditions at 40° C. for 30 minutes. (d) Stability against temperature: Stable up to 45°C under conditions of pH 6.5 and heating for 30 minutes. (e) Range of optimum temperature for action: The optimum temperature for action is around 50°C. (f) Inactivation conditions: pH 5.0 and 1 under treatment conditions of 40°C and 30 minutes.
At 0, it is completely inactivated. Furthermore, when treated at pH 6.5 for 30 minutes, the activity is completely inactivated at 55°C. (g) A microorganism belonging to the genus Bacillus that has an optimum pH for growth on the alkaline side and has the ability to produce β-mannosidase with a molecular weight of 63,000 ± 3,000 by gel filtration method is cultured, and the β-mannosidase is A method for producing a novel intramicrobial β-mannosidase, which comprises producing and accumulating it in the body and collecting it. (4) The method for producing intracellular β-mannosidase according to claim 3, wherein the culture is carried out aerobically at a temperature within the range of 30 to 45°C. (5) Claim 3 or 4, characterized in that the pH of the culture solution is within the range of 7.5 to 11.5.
A method for producing intracellular β-mannosidase as described in 2. (6) Any one of claims 3 to 5, wherein the microorganism is a microorganism deposited as FERMP-8860 (AS-440) with the Institute of Microbial Technology, Agency of Industrial Science and Technology. The method for producing β-mannosidase described in .